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Direct lineage conversion

In October 2012 Shinya Yamanaka and John Gurdon received “the call” that is every scientist’s dream. The Nobel Prize committee in Stockholm voted to award them that year’s Nobel Prize in Physiology or Medicine for their work demonstrating “that mature cells can be reprogrammed to become pluripotent.”
For those in the stem cell field, the news could hardly have been called surprising. Recognition of Gurdon’s classic experiment demonstrating that adult frog cell nuclei could be used to grow tadpoles, was decades in coming. Yamanaka’s work, though more recent, has been practically earth-shattering. Nearly 5,000 research papers to date have cited his seminal 2006 paper on mouse induced pluripotent stem (iPS) cells, according to the Web of Knowledge. [1] That paper established the concept that the developmental clocks of terminally differentiated cells like fibroblasts could be “rewound” (or reprogrammed) to an embryonic stem cell-like state via the transient expression of a handful of transcription factors, which work their magic via widespread alteration of the cellular epigenome. Like ES cells, the resulting cells can then be differentiated into any other cell type in the body, an observation with profound implications for researchers, drug developers, and clinicians, who recognized that neurons derived from Parkinson patient iPS cells, for instance, could be used to screen candidate therapeutics or could potentially be genetically repaired for transplant into patients.
Yet it turns out researchers don’t necessarily need iPS cells to produce those cells after all. As early as 1987, Robert Davis et al. showed that expression of MyoD could directly convert fibroblasts into myoblasts. [2] In 2008 Harvard Stem Cell Institute researcher Doug Melton and colleagues demonstrated conversion of pancreatic exocrine cells to insulin-secreting beta-cells in mice by co-expressing three transcription factors, Ngn3, Pdx1, and Mafa. [3]
Yet these and other examples all involve relatively closely related lineages, says Marius Wernig, Assistant Professor of Pathology at the Stanford University School of Medicine; the question was, could more distant lineages be interconverted?
In 2010 Wernig published a Nature article showing that fibroblasts (a mesoderm-derived cell type) could be directly, rapidly, and efficiently converted to neurons (ectoderm) -- or more precisely, “induced neuronal” (iN) cells -- without passing through a pluripotent intermediate. [4] “Earlier, only very closely related lineages could be converted,” he explains. “Now we showed this huge jump was possible. That suggests that if you know the right conditions and transcription factors, probably you can convert anything into anything.”
In this case, nearly 20% of fibroblasts were converted to neurons in some experiments, in just 12 days. By contrast, iPS cell generation is highly inefficient and takes weeks to months, after which individual lines must then be characterized.
Still, Wernig’s technique stems intellectually from Yamanaka. Starting with 19 neurally restricted transcription factors, the team identified three (Ascl1, Brn2, and Myt1l) that, when co-expressed in mouse embryonic fibroblasts, caused the cells to morph into neurons. “Certainly, we were inspired by Yamanaka,” Wernig says. Writing in a “News & Views” article accompanying that paper, UCSF stem cell researchers Cory Nicholas and Arnold Kriegstein described the discovery as “a feat of biological wizardry.” [5]
Today, several groups have extended that wizardry, showing that human cells may also be “trans-differentiated,” as can cells other than fibroblasts. (For a recent review, see ref. [6].) They also have devised alternative strategies to induce the trans-differentiation process. In either case, trans-differentiation (or “direct lineage conversion”) is proving to be a versatile and robust process — one that, like iPS cell creation, has applications in the research, drug development, and clinical arenas.

TWO METHODS OF TRANSDIFFERENTIATION
Wernig’s original study used neural transcription factors to turn fibroblasts into neurons. But neurons are terminally differentiated and post-mitotic. That is, they don’t divide and cannot be expanded in culture. That can be a problem for drug developers who might wish to seed thousands of wells for screening assays, not to mention clinicians who hope to use such cells for cell-based therapies.
To circumvent those issues, Wernig’s team developed a modified method that generates replicative, tripotent “induced neural precursor cells” (iNPCs) that give rise to neurons, astrocytes, or oligodendrocytes. [7] Unlike the original procedure, Wernig’s new method uses transcription factors expressed throughout neural differentiation; starting with 11 such proteins, he narrowed the list down to three: Brn2, Sox2, and FoxG1. When added to mouse fibroblasts, these three proteins produce tripotent cells at a frequency of about 11.5% in 21 days. [8]
An alternative strategy, advanced by Senior Investigator Sheng Ding of the Gladstone Institute of Cardiovascular Disease at the University of California, San Francisco, repurposes the Yamanaka pluripotency factors and specially formulated growth media to transform fibroblasts into neuronal precursors. [9]
“That is very different from the existing paradigm,” Ding says. “The existing paradigm is [that] for any cell type you want to make, you use specific [transcription] factors for that lineage.” In contrast, his team used universal pluripotency factors, but for a limited time of three or four days, and coupled them with a different set of growth factors, to accomplish the transdifferentiation process.
Conceptually, says Ding, one can envision his process as a partial rewinding of the fibroblasts’ cellular clock, followed by redifferentiation along the neuronal pathway. But the reality is “more complex than that,” he says.
“The Yamanaka factors and signaling factors do not act separately, they act together,” he explains. Just as two physical forces can combine to form a single vector, like the concerted forces of vertical lift and rotational centripetal force that act upon a banking airplane wing, the transcription factors and growth factors in Ding’s protocol cooperate to convert fibroblasts into neurons.
Ding has also shown that transdifferentiation from human fibroblasts to functional neurons can be accomplished using two transcription factors (Myt1l and Brn2) plus the microRNA miR-124. [10] That observation, Ding says, was “surprising,” because “we don’t understand the underlying mechanisms.” MicroRNAs function by an entirely different mechanism than transcription factors, yet appear to produce the same effect. “So why miRNA be able to induce reprogramming, is not understood,” he says.

IT’S NOT ALL ABOUT NEURONS
Lineage conversion processes would have limited application if they could be used only to convert fibroblasts into neurons. As it turns out, they are more broadly applicable than that. A few highlights:
August 2010: Deepak Srivastava and colleagues at the Gladstone Institute (not including Ding) convert murine fibroblasts into cardiomyocytes using the transcription factors, Gata4, Mef2c, and Tbx5. [11]
March 2011: Ding’s team transdifferentiates mouse fibroblasts into cardiomyocytes in about three weeks using the Yamanaka factors and chemical defined media. [12]
July 2011: Chinese and Japanese research teams independently report the direct conversion of mouse fibroblasts into “hepatocyte-like cells” (liver cells) using transcription factor cocktails comprising HNF4alpha plus Foxa1, Foxa2, or Foxa3 [13] or Gata4, HNF1alpha, Foxa3, and RNAi-mediated inactivation of p19Arf. [14]
October 2011: Wernig’s team converts hepatocytes to functional iN cells. [15]
December 2012: Researchers led by Juan Carlos Izpisua Belmonte at the Salk Institute for Biological Studies, use a “partial reversion” process, similar in concept to Ding’s, to convert human fibroblasts to “angioblast-like” progenitor cells (that is, cells can can differentiate into endothelial and smooth muscle lineages). [16]
December 2012: Researchers in China use pluripotency factors, microRNAs, and small molecule inhibitors to convert cells in human urine to neural progenitors. [17]
February 2013: Harvard researchers Caroline Rouaux and Paolo Arlotta overexpress the Fezf2 transcription factor in one subtype of post-mitotic excitatory neurons (callosal projection neurons) to convert them into another subtype (corticofugal neurons) in vivo. [18] These data, the authors say, “indicate that critical features of neuronal subtype-specific identity, including the acquisition of a new molecular identity and the ability to extend axons to a new target, can still be changed post-mitotically, even several days after initial fate-specification.”

Even iPS cells themselves can be differentiated via the transcription factor-addition method. “What we have seen is when you convert iPS cells into neurons the way we convert fibroblasts to neurons, we get neurons in record time,” Wernig says — in as few as two or three weeks. “These cells look much more mature in a given time point compared to standard differentiation protocols, but also more homogeneous. So that is a literal marriage of the two techniques.”

DIFFERENT PROTOCOLS, DIFFERENT APPLICATIONS
According to Ignacio Sancho-Martinez, a senior postdoc in Belmonte’s group at the Salk Institute and coauthor of the group’s 2012 paper, lineage-converted cells provide certain advantages over iPS cells, though both have applications. The process is far faster, for one thing. It also produces fewer mutations, and theoretically eliminates the risk of teratoma formation that is always present when dealing with pluripotent cells.
“You don’t have the safety issues you have with iPS cells,” he says, though he adds, “But you have to test that in the clinic.”
Another benefit, Wernig says, is that it is easier to use direct differentiation when handling multiple patients, because the standard when producing iPS cells is to characterize a minimum of three iPS lines per sample.
“If you want to compare 50 patients with another set of 50 patients and 50 controls, that is very difficult to do with iPS cells, because you have to convert … at least 450 clones.” For such studies, he says, “the direct approach is probably better.” On the other hand, other drug screening applications are probably more easily accomplished using iPS cells, as these are more proliferative than even iNPCs.
Still, says William Lowry, the Maria Rowena Ross Professor of Molecular, Cell and Developmental Biology at the University of California, Los Angeles, there are at least two significant unresolved questions regarding transdifferentiated cells that must be answered before they can be considered a viable alternative to iPS cells, at least clinically: How does the reprogramming process happen — does it work, as the Yamanaka process does, via changes in the epigenetic landscape, for instance? And how similar is one transdifferentiated cell to another and to cells in vivo. “It’s pretty easy to define a pluripotent cell in vitro,” he says. “It’s harder when thinking of the dozens or hundreds of different types of neurons in the central nervous system.”

Comments

Oh My God! 5000 papers in all. Awesome that is. Even if I spend my whole life preparing presentation papers even 500 would be impossible for me. How do people manage doing it will always be a suspense for me.

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Copyright 2013 by the Massachusetts General Hospital. Some sections copyright 2008-2009 by The President and Fellows of Harvard College.